Prodrugs of a JAK inhibitor compound for treatment of gastrointestinal inflammatory disease

ABSTRACT

The invention provides compounds which are prodrugs of a JAK inhibitor agent for the targeted delivery of the JAK inhibitor to the gastrointestinal tract of a mammal. The invention also provides pharmaceutical compositions comprising the compounds, methods of using the compounds to treat gastrointestinal inflammatory diseases, and processes and intermediates useful for preparing the compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/259,273, filed on Nov. 24, 2015; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention is directed to compounds designed for targeted delivery ofa JAK inhibitor to the gastrointestinal tract. The invention is alsodirected to pharmaceutical compositions comprising such compounds,methods of using such compounds to treat gastrointestinal inflammatorydiseases, and processes and intermediates useful for preparing suchcompounds.

State of the Art

Inflammatory bowel disease, which primarily includes ulcerative colitisand Crohn's disease, involves chronic inflammation of all or part of thegastrointestinal tract. Ulcerative colitis is characterized byinflammation and ulceration of the mucosal layer of the rectum and thelarge intestine while Crohn's disease largely involves the ileum but canoccur anywhere along the intestinal tract. Common symptoms includediarrhea, bloody stools, and abdominal pain. The clinical course ofulcerative colitis is intermittent, marked by alternating periods ofexacerbation and remission. Incidence seems to be greater in developedthan in developing countries. An estimated 1.3 million people in majorindustrialized countries suffer from ulcerative colitis and the numbersare expected to increase along with population growth. Patients withulcerative colitis are at an increased risk of developing colorectalcancer. (e.g., Danese et al. N Engl J Med, 2011, 365, 1713-1725). Inaddition, an estimated 1 million people in industrialized countriessuffer from Crohn's disease.

Although there exists a variety of therapeutic options to promote andmaintain remission of ulcerative colitis (UC) in patients, none isideal. Sulfasalazine-related treatments are often effective in mild UC,but much less so in moderate to severe disease. Corticosteroids areoften used to provide rapid induction of remission in patients withmoderate to severe UC. However, chronic use of steroids to maintainremission is discouraged due to their association with longer termadverse effects (e.g., osteoporosis and fractures, infections,cataracts, slower wound healing and suppression of adrenal gland hormoneproduction). Systemic immunosuppressants such as azathioprine,cyclosporine and methotrexate have a slow onset and modest efficacy inmoderate to severe UC patients, but prolonged use can be problematic dueto consequences of long-term systemic immunosuppression (e.g., increasedrisk of infections and lymphoma). Anti-TNFα antibodies (e.g., infliximaband adalimumab), while expensive and requiring subcutaneous orintravenous administration, are efficacious in approximately 60 to 70%of UC patients with moderate to severe disease. However, up to one thirdof patients fail to respond adequately, while another third of initialresponders develop tolerance over a few weeks (Allez et al., J Crohn'sColitis, 2010, 4, 355-366; Rutgeerts et al., N Engl J Med, 2005, 353,2462-2476). The most recently approved UC therapy, vedolizumab, ananti-α₄β₇ integrin antibody, is efficacious in moderate to severe UCpatients although its parenteral route is suboptimal, and theconsequences of long-term immunosuppression via this mechanism remain tobe determined. Despite existing therapeutic options, about 10 to 20% ofUC patients still require colectomy within 10 years of diagnosis(Targownik et al., Am J Gastroenterol, 2012, 107, 1228-1235). It isclear there remains an unmet medical need for an effective therapy topromote and maintain remission of moderate to severe UC without thesafety concerns resulting from chronic, systemic immunosuppression.

While the mechanism underlying ulcerative colitis is not completelyunderstood, it is believed that environmental factors in geneticallysusceptible individuals evoke an inappropriate (excessive) reaction bythe immune system to gut microbiota, resulting in colonic inflammation,tissue damage, and the associated symptoms characteristic of thedisease.

Although the precise pathogenesis of UC is unclear, it is apparent thatproinflammatory cytokines play a pivotal role in the immunologicalresponse (Strober et al., Gastroenterol, 2011, 140, 1756-1767). Many ofthe proinflammatory cytokines most commonly elevated in UC (e.g., IL-4,IL-6, IL-13, IL-15, IL-23, IL-24, IFNγ and leptin), rely on the JAKfamily of tyrosine kinases (i.e., JAK1, JAK2, JAK3 and Tyk2) for signaltransduction. Ligand binding to a cytokine receptor triggersautophosphorylation of its associated JAK, which in turn results inphosphorylation of a signal transducer and activator of transduction(STAT) protein. Different STATs form hetero- or homodimers and promotetranscription of their target genes in the cell nucleus to regulatefunctions such as cell growth, differentiation and death (Clark et al.,J Med Chem, 2014, 57, 5023-5038).

Inhibition of the family of JAK enzymes could inhibit signaling of manykey proinflammatory cytokines. Thus JAK inhibitors are likely to beuseful in the treatment of ulcerative colitis and other inflammatorydiseases such as Crohn's disease, allergic rhinitis, asthma, and chronicobstructive pulmonary disease (COPD). However, due to the modulatingeffect of the JAK/STAT pathway on the immune system, systemic exposureto JAK inhibitors may have an adverse systemic immunosuppresive effect.

Tofacitinib citrate (Xeljanz®), an oral, systemically available, pan-JAKinhibitor, was approved in the United States in November, 2012 to treatadults with moderately to severely active rheumatoid arthritis who havehad an inadequate response to, or who are intolerant of, methotrexate.While demonstrating superior efficacy at higher doses in clinicalstudies, tofacitinib was only approved at a 5 mg twice daily (BID) dosebased on dose-limiting, systemically-mediated, adverse events (e.g.,elevated cholesterol, increased rate of opportunistic infections,neutropenia, lymphocytopenia, lymphoma and solid tumors). The drugcarries a boxed warning in the US detailing the safety risks and wasdeclined approval in Europe based on ‘significant and unresolvedconcerns’ about the overall safety profile. Tofacitinib is under activedevelopment for UC having demonstrated a clinical response in a Phase 2(8 week) UC trial (Sandborn et al., N Engl J Med, 2011, 365, 1713-1725),particularly at the 10 mg and 15 mg BID dose (See also. Panes et al.,BMC Gastroenterol, 2015, 15, 14), doses not currently approved for anyindication. The sponsor has also reported a greater proportion ofpatients receiving tofacitinib 10 mg BID as compared to placebo were inremission in a Phase 3 (8 week) UC induction trial and also for patientsreceiving tofacitinib 5 mg and 10 mg BID in a Phase 3 (52 week) UCmaintenance trial.

For the treatment of ulcerative colitis and other gastrointestinalinflammatory diseases, it would be desirable to provide a compound thaton oral administration achieves sufficiently high exposure oftofacitinib in the gastrointestinal tract to optimize clinical efficacywhile avoiding systemic dose-limiting systemic exposure.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides novelglucuronide-containing prodrugs of the JAK inhibitor tofacitinib. Suchprodrugs take advantage of the gut microbiome which contains or producesan abundance of β-glucuronidase which selectively cleaves theglucuronide-containing prodrug moiety to trigger release of tofacitinibin the gastrointestinal tract, in particular in the colon.

Surprisingly, the glucuronide-containing prodrugs of the presentinvention are sufficiently stable to be isolated, formulated in apharmaceutical composition and administered to a patient in need oftreatment. However, they are also sufficiently labile so as to becleaved by β-glucuronidase and further break-down to efficiently releasetofacitinib. In contrast, when the glucuronide-containing prodrug moietyemployed in the present invention was attached to certain other drugsknown or potentially useful for treating UC, the drugs were not releasedon contact with β-glucuronidase (as described more fully herein below).Accordingly, the glucuronide-containing prodrugs of the presentinvention are particularly useful for delivering and releasingtofacitinib.

In one aspect, the present invention relates to a compound of formula(I):

wherein

n is 0, 1 or 2;

R¹ is selected from hydrogen, C₁₋₄ alkyl, C₁₋₃ alkoxy, amino, nitro,halo, cyano, hydroxy, and trifluromethyl;

each R², when present, is independently selected from C₁₋₄ alkyl, C₁₋₃alkoxy, amino, nitro, halo, cyano, hydroxyl, and trifluromethyl;

R³ is hydrogen, methyl or ethyl;

R⁴ is hydrogen, methyl or ethyl;

or a pharmaceutically-acceptable salt thereof.

In another aspect, the present invention relates to a compound offormula (II):

wherein

R¹ is selected from hydrogen, C₁₋₄ alkyl, C₁₋₃ alkoxy, amino, nitro,halo, cyano, hydroxy, and trifluromethyl;

or a pharmaceutically-acceptable salt thereof.

In one embodiment, the invention provides a compound of formula (II)wherein R¹ is selected from hydrogen, methyl, methoxy, amino, nitro, andchloro, or a pharmaceutically-acceptable salt thereof.

In another aspect, the present invention relates to a compound offormula 1:

or a pharmaceutically acceptable salt thereof.

The compound of formula 1 has demonstrated low oral bioavailability androbust release of tofacitinib (2)

in vivo upon oral administration in preclinical species resulting in amarked increase in the ratio of colon exposure to plasma exposurerelative to that obtained on oral dosing of tofacitinib itself.

In one embodiment, the compound of formula 1 produces tofacitinib or asalt thereof, upon contact with β-glucuronidase.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising a pharmaceutically acceptable-carrier and acompound of formula (I), (II) or 1, or a pharmaceutically acceptablesalt thereof; or any specific embodiments thereof described herein.

In another aspect, the present invention relates to a method of treatinga gastrointestinal inflammatory disease in a mammal, the methodcomprising administering to the mammal a pharmaceutical compositioncomprising a pharmaceutically acceptable-carrier and a compound offormula (I), (II) or 1, or a pharmaceutically acceptable salt thereof;or any specific embodiments thereof described herein.

In one embodiment, the gastrointestinal inflammatory disease isulcerative colitis. In another embodiment, the gastrointestinalinflammatory disease is Crohn's disease. And in another embodiment, thegastrointestinal inflammatory disease is colitis associated with immunecheckpoint inhibitor therapy.

In another aspect, the present invention relates to a method ofdelivering tofacitinib to the gastrointestinal tract of a mammal, inparticular, to the colon, the method comprising orally administering tothe mammal a glucuronide-containing prodrug of tofacitinib which prodrugis cleaved by β-glucuronidase in the gastrointestinal tract to releasetofacitinib.

In separate and distinct embodiments, the glucuronide-containing prodrugof tofacitinib is a compound of formula (I), (II) or 1, or apharmaceutically acceptable salt thereof; or any specific embodimentsthereof described herein.

In another aspect, the present invention relates to a process forpreparing a compound of formula (I) or a pharmaceutically acceptablesalt thereof, the process comprising deprotecting a compound of formula(I-A):

or a salt thereof; wherein R¹, R², R³, R⁴ and n are as defined herein;each PG^(a) is independently a hydroxyl protecting group; and PG^(b) isa carboxyl protecting group; to provide a compound of formula (I) or apharmaceutically acceptable salt thereof.

In one embodiment of this process, R¹ is nitro; R³ and R⁴ are methyl;each PG^(a) is acetyl; PG^(b) is methyl; and n is 0.

In another aspect, the present invention relates to a compound offormula (I-A), or a salt thereof; or any specific embodiments thereofdescribed herein.

In another aspect, the present invention relates to a process forpreparing a compound of formula 1, or a pharmaceutically acceptable saltthereof, the process comprising:

(a) reacting a compound of formula 12′

or a salt thereof; wherein each PG^(a) is independently a hydroxylprotecting group, with a compound of formula 13

to provide a compound of formula 14′:

and

(b) deprotecting the compound of formula 14′ to provide the compound offormula 1 or a pharmaceutically acceptable salt thereof.

In one embodiment of this process, PG^(a) is acetyl.

In separate and distinct aspects, the present invention also relates toa compound of formula 13, or a salt thereof; and a compound of formula14′ or a salt thereof, or any specific embodiments thereof describedherein.

In separate and distinct aspects, the present invention also relates toother synthetic processes and intermediates described herein, which areuseful for preparing the compounds of the invention.

In separate and distinct aspects, the present invention also relates toa compound of formula (I), (II) or 1, or a pharmaceutically acceptablesalt thereof; or any specific embodiments thereof described herein; foruse in medical therapy; or for use in the manufacture of a medicament ora formulation. In one embodiment, the medicament or formulation is fortreating a gastrointestinal inflammatory disease in a mammal.

Other aspects and embodiments of this invention are disclosed herein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the concentration of active metabolites, tofacitiniband compounds C-1M and C-2M, respectively as a function of time as aresult of incubation of the compound of the invention (compound 1) andof comparison compounds C-1 and C-2 with rat colon content.

DETAILED DESCRIPTION OF THE INVENTION

Among other aspects, the invention provides glucuronide prodrugs of theJAK kinase inhibitor tofacitinib, pharmaceutically-acceptable saltsthereof, and intermediates for the preparation thereof.

Chemical structures are named herein according to IUPAC conventions asimplemented in ChemDraw software (PerkinElmer, Inc., Cambridge, Mass.).According to the convention, the compound of formula 1 may be identifiedas:

-   (2S,3S,4S,5R,6S)-6-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic    acid,

while tofacitinib (2) may be identified as3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile.

The compounds of the invention contains multiple chiral centers. Thedepiction or naming of a particular stereoisomer means the indicatedstereocenter has the designated stereochemistry with the understandingthat minor amounts of other stereoisomers may also be present unlessotherwise indicated, provided that the utility of the depicted or namedcompound is not eliminated by the presence of another stereoisomer.

Definitions

When describing this invention including its various aspects andembodiments, the following terms have the following meanings, unlessotherwise indicated.

The singular terms “a,” “an” and “the” include the corresponding pluralterms unless the context of use clearly dictates otherwise.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to about 10 carbonatoms. Representative alkyl groups include, by way of example, methyl,ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, n-hexyl, 2,2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl,2-ethylbutyl, 2,2-dimethylpentyl, 2-propylpentyl, and the like.

When a specific number of carbon atoms are intended for a particularterm, the number of carbon atoms is shown preceding the term. Forexample, the term “C₁₋₃ alkyl” means an alkyl group having from 1 to 3carbon atoms wherein the carbon atoms are in any chemically-acceptableconfiguration, including linear or branched configurations.

The term “alkoxy” means the monovalent group —O-alkyl, where alkyl isdefined as above. Representative alkoxy groups include, by way ofexample, methoxy, ethoxy, propoxy, butoxy, and the like.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treatment” as used herein means the treatment of a disease,disorder, or medical condition (such as a gastrointestinal inflammatorydisease), in a patient, such as a mammal (particularly a human) whichincludes one or more of the following:

(a) preventing the disease, disorder, or medical condition fromoccurring, i.e., preventing the reoccurrence of the disease or medicalcondition or prophylactic treatment of a patient that is pre-disposed tothe disease or medical condition;

(b) ameliorating the disease, disorder, or medical condition, i.e.,eliminating or causing regression of the disease, disorder, or medicalcondition in a patient, including counteracting the effects of othertherapeutic agents;

(c) suppressing the disease, disorder, or medical condition, i.e.,slowing or arresting the development of the disease, disorder, ormedical condition in a patient; or

(d) alleviating the symptoms of the disease, disorder, or medicalcondition in a patient.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts derived fromacids include salts of acetic, ascorbic, benzenesulfonic, benzoic,camphorsulfonic, citric, ethanesulfonic, edisylic, fumaric, gentisic,gluconic, glucoronic, glutamic, hippuric, hydrobromic, hydrochloric,isethionic, lactic, lactobionic, maleic, malic, mandelic,methanesulfonic, mucic, naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

Salts derived from pharmaceutically-acceptable inorganic bases includeammonium, calcium, magnesium, potassium, sodium, and zinc, and the like.Salts derived from pharmaceutically-acceptable organic bases includesalts of arginine, choline, glucamine, lysine, benethamine, benzathine,betaine, 2-dimethylaminoethanol, 2-diethylaminoethanol, hydrabamine,morpholine, tromethamine, diethanolamine, ethanolamine, ethylenediamine,triethanolamine, 1H-imidazole, piperazine, and the like.

The term “salt thereof”, as used herein, means an ionic compound inwhich a form of a compound of formula (I) is either the anion or cationof the ionic compound. For example, the anion of the ionic compound canbe a carboxylate anion that is a deprotonated form of a compound offormula (I). The cation can be a protonated form of a compound offormula (I), i.e. a form where an amino group has been protonated by anacid. Typically, the salt is a pharmaceutically acceptable salt,although this is not required for salts of intermediate compounds thatare not intended for administration to a patient.

Neutral compounds of formula (I) may optionally take the form of azwitterion, where the term “zwitterion” means a neutral molecule withboth positive and negative electrical charges.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesired reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, alkylgroups, such as methyl, ethyl, and tert-butyl; allyl groups; acylgroups, for example alkanoyl groups, such as acetyl; arylmethyl groups,such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), anddiphenylmethyl (benzhydryl, DPM); silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBS); and the like.

The term “carboxyl-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxyl group. Representativecarboxyl-protecting groups include, but are not limited to, alkylgroups, such as methyl, ethyl, tert-butyl, and the like; arylmethylgroups, such as benzyl, 4-nitrobenzyl, 4-methoxybenzyl and the like;thiol groups, such as —S-tert-butyl and the like; silyl groups, such astrimethylsilyl, tert-butyldimethylsilyl and the like; oxazolines; andthe like.

All other terms used herein are intended to have their ordinary meaningas understood by persons having ordinary skill in the art to which theypertain.

Representative Embodiments and Subgeneric Groupings

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of thisinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of this invention.

In one embodiment, R¹ is hydrogen, C₁₋₄ alkyl, C₁₋₃ alkoxy, amino,nitro, halo, cyano, hydroxy, or trifluromethyl. In another embodiment,R¹ is hydrogen, C₁₋₄ alkyl, C₁₋₃ alkoxy, amino, nitro, or chloro. Inanother embodiment, R¹ is hydrogen, methyl, methoxy, amino, nitro orchloro. In a particular embodiment, R¹ is hydrogen. In anotherparticular embodiment, R¹ is nitro.

In one embodiment, n is 0. In another embodiment, n is 1. In anotherembodiment, n is 2.

When n is 1, in one embodiment, R² is C₁₋₄ alkyl, C₁₋₃ alkoxy, amino,nitro, halo, cyano, hydroxyl, or trifluromethyl. In another embodiment,R² is C₁₋₄ alkyl, C₁₋₃ alkoxy, amino, nitro, fluoro or chloro. Inanother embodiment, R² is methyl, methoxy, amino, nitro, fluoro orchloro. In a particular embodiment, R² is fluoro.

When n is 1, the R² substituent may be in any available position of thephenyl ring to which R² is attached. In one embodiment, R² is ortho toR¹. In another embodiment, R² is meta to R¹. In another embodiment, R²is para to R¹.

When n is 2, in one embodiment, each R² is independently C₁₋₄ alkyl,C₁₋₃ alkoxy, amino, nitro, halo, cyano, hydroxyl, or trifluromethyl. Inanother embodiment, each R² is independently C₁₋₄ alkyl, C₁₋₃ alkoxy,amino, nitro, fluoro or chloro. In another embodiment, each R² isindependently methyl, methoxy, amino, nitro, fluoro or chloro. In aparticular embodiment, each R² is fluoro.

When n is 2, the R² substituents may be in any available position of thephenyl ring to which R² is attached. In one embodiment, the R²substituents are ortho and meta to R¹. In another embodiment, the R²substituents are ortho and para to R¹. In another embodiment, the R²substituents are meta and para to R¹.

In one embodiment, R³ is hydrogen. In another embodiment, R³ is methyl.In another embodiment, R³ is ethyl.

In one embodiment, R⁴ is hydrogen. In another embodiment, R⁴ is methyl.In another embodiment, R⁴ is ethyl.

In one embodiment, both R³ and R⁴ are methyl. In another embodiment, oneof R³ and R⁴ is hydrogen and the other is methyl.

In one embodiment, n is 0; R¹ is hydrogen, methyl, methoxy, amino, nitroor chloro; R³ is methyl; and R⁴ is methyl.

In another embodiment, n is 0; R¹ is hydrogen, methyl, methoxy, amino,nitro or chloro; R³ is hydrogen; and R⁴ is methyl.

In another embodiment, n is 0; R¹ is hydrogen, methyl, methoxy, amino,nitro or chloro; R³ is methyl; and R⁴ is hydrogen.

In another embodiment, n is 0; R¹ is hydrogen, methyl, methoxy, amino,nitro or chloro; R³ is ethyl; and R⁴ is ethyl.

In another embodiment, n is 1; R¹ is hydrogen, methyl, methoxy, amino,nitro or chloro; R² is methyl, methoxy, amino, nitro, fluoro or chloro;R³ is methyl; and R⁴ is methyl.

In another embodiment, n is 1; R¹ is hydrogen, methyl, methoxy, amino,nitro or chloro; R² is methyl, methoxy, amino, nitro, fluoro or chloro;R³ is hydrogen; and R⁴ is methyl.

In another embodiment, n is 1; R¹ is hydrogen, methyl, methoxy, amino,nitro or chloro; R² is methyl, methoxy, amino, nitro, fluoro or chloro;R³ is methyl; and R⁴ is hydrogen.

Synthetic Procedures

Compounds of formula (I) may be prepared according to the syntheticapproach described in detail in the appended examples. As illustrated inScheme 1 specifically for the preparation of the compound of formula 1,the key step of the synthesis is the formation of the urea linkagebetween tofacitinib and a protected form of the glucuronide prodrugmoiety 12′. In Scheme 1, PG^(a) represents a hydroxyl protecting group,preferably allyl or acetyl, although other hydroxyl protecting group mayalso be used including a silyl protecting group such astert-butyldimethylsilyl.

Formation of this key urea linkage initially focused on usingtofacitinib as the nucleophile; however this bond forming step was foundto be inconsistent as reaction of tofacitinib with a range of differentelectrophiles only led to formation of the desired product in lowyields. It was therefore deemed necessary to switch the role of the twopartners and make a tofacitinib derivative the electrophilic reactingpartner, and the glucuronide prodrug moiety the nucleophile. Aftersurveying many reagents, it was determined that tofacitinib could bederivatized and rendered electrophilic when it was joined to a reactivepara-nitrophenyl or pentafluorophenyl moiety via a carbamate linkage.For example, para-nitrophenyl chloroformate, bis(4-nitrophenyl)carbonate, or bis(pentafluorophenyl) carbonate reagents were able toachieve the desired balance of being reactive enough to affect the bondformation with tofacitinib, while also providing an intermediate whichwas not so reactive that it decomposes before reaction with theglucuronide species. The resulting protected intermediate 14′ isdeprotected, for example when PG^(a) is an acetate group, with lithiumhydroxide in a subsequent step to provide the compound of formula 1.

Accordingly, in one aspect, the invention provides a process ofpreparing compound 1, the process comprising (a) reacting a protectedglucuronide prodrug moiety 12′ with an electrophilic tofacitinibderivative 13 to provide compound 14′, and (b) deprotecting compound 14′to provide the compound of formula 1. In a further aspect, the inventionprovides the electrophilic tofacitinib compound 13.

The action of a β-glucuronidase enzyme on the prodrugs of the inventionis illustrated for the compound of formula 1. As shown in Scheme 2, uponaction of a β-glucuronidase enzyme on the compound of formula 1,tofacitinib is released by a multistep process:

In the initial step, the β-glucuronidase enzyme cleaves the glycosidicbond of the compound of formula 1, causing glucuronic acid (a) to bereleased and the formation of an aglycone intermediate (b). The aglyconespontaneously decomposes to afford a quinone methide species (c) whichcan be trapped with water, and a transient carbamic acid which losescarbon dioxide to afford a diamine (d). Intramolecular cyclization ofthe diamine leads to the formation of an imidazolidinone derivative (e)and release of tofacitinib.

As described in the experimental section below, the conversion of thecompound of formula 1 to tofacitinib via the intermediate stepsillustrated in Scheme 2 has been observed in incubations with purifiedβ-glucuronidase (Assay 2) and with freshly prepared rat colon contenthomogenate (Assay 3). In these latter experiments, the concentrations ofthe compound of formula 1, the aglycone intermediate b, the diamineintermediate d, and tofacitinib were monitored as a function of time.The rapid disappearance of the compound of formula 1 was accompanied bythe rapid and transient formation of the aglycone b and the slower,rate-determining appearance of the diamine d and the ultimate activemetabolite tofacitinib.

The present investigators have determined that the multistepdecomposition of the glucuronide prodrug compound starting with thebacterial β-glucuronidase enzymatic cleavage of the glycosidic bond tobeneficially deliver a desired active moiety directly to the site ofaction in the colon depends sensitively on the nature of the linkage andon the specific active moiety. Mesalamine, 5-aminosalicylic acid (5-ASA)(compound C-1M) is an older agent, long used for the treatment of mildto moderate ulcerative colitis. However, the present glucuronide prodrugapproach does not appear to be applicable for delivering 5-ASA directlyto the colon. The glucuronide prodrug of 5-ASA (compound C-1)

was incubated with rat colon content homogenate in an analogousexperiment to that noted above. While a transient aglycone analogous toaglycone b in Scheme 2 and a slower appearance of a diamine analogous tod were observed on a time scale similar to that for the compound offormula 1, no evidence of active metabolite 5-ASA could be found.

A recent publication US2013/0109720 disclosed2-(5-fluoro-4-methylpyridin-3-yl)-5-(4-methyl-6-(methylsulfonyl)pyridin-3-yl)-1H-indole(compound C-2M) as a calcium release-activated calcium channel (CRAC)inhibitor. CRAC inhibitors are also believed to be useful for thetreatment of inflammatory diseases. However, the present glucuronideprodrug approach also does not appear to be applicable for the targeteddelivery of this CRAC inhibitor. The breakdown of the CRAC inhibitorprodrug C-2

was also studied in rat colon content homogenate with results similar tothose obtained for compound C-1. While intermediate breakdown productswere observed, no evidence of release of the active CRAC inhibitor C-2Mcould be found.

The present evidence suggests the prodrugs of the invention are uniquelysuited to take advantage of a bacterial β-glucuronidase-initiatedbreakdown mechanism to release tofacitinib in the colon.

Pharmaceutical Compositions

The compounds of the invention and pharmaceutically-acceptable saltsthereof are typically used in the form of a pharmaceutical compositionor formulation. Accordingly, in one of its compositions aspects, theinvention is directed to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a compound offormula (I), (II), or 1, or a pharmaceutically-acceptable salt thereof.Optionally, such pharmaceutical compositions may contain othertherapeutic and/or formulating agents if desired.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present invention.Those skilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.1to about 95% by weight of the compound of formula (I); including fromabout 5 to about 70% by weight; such as from about 10 to about 60% byweight of the compound of formula (I).

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending a compound of the invention with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of the present compounds calculatedto produce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like, or unit packages suitablefor parenteral administration.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for oral administration. Suitable pharmaceutical compositionsfor oral administration may be in the form of capsules, tablets, pills,lozenges, cachets, dragees, powders, granules; or as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil liquid emulsion; or as an elixir or syrup; and the like;each containing a predetermined amount of a compound of the presentinvention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise a compound of the invention andone or more pharmaceutically-acceptable carriers, such as sodium citrateor dicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, dicalcium phosphate, sucrose,glucose, mannitol, and/or silicic acid; binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as crosscarmellose sodium, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents, such ascitric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid,phosphoric acid, and the like. Coating agents for tablets, capsules,pills and like, include those used for enteric coatings, such ascellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, methacrylic acid, methacrylic acid estercopolymers, cellulose acetate trimellitate, carboxymethyl ethylcellulose, hydroxypropyl methyl cellulose acetate succinate, and thelike.

Pharmaceutical compositions of the invention may also be formulated toprovide slow or controlled release of the active agent using, by way ofexample, hydroxypropyl methyl cellulose in varying proportions; or otherpolymer matrices, liposomes and/or microspheres. In addition, thepharmaceutical compositions of the invention may optionally containopacifying agents and may be formulated so that they release the activeingredient only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active agent can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (esp., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), oleic acid, glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Alternatively, certain liquid formulations can be converted,for example, by spray drying, to a powder, which is used to preparesolid dosage forms by conventional procedures.

Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Tablet Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof is dry blended with microcrystalline cellulose, polyvinylpyrrolidone, and croscarmellose sodium in a ratio of 4:5:1:1 andcompressed into tablets to provide a unit dosage of, for example, 4 mg,10 mg or 20 mg active agent per tablet.

Tablet Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof (40 g) is thoroughly blended with microcrystalline cellulose(445 g), silicon dioxide fumed (10 g), and stearic acid (5 g). Themixture is then compressed on a tablet press to form tablets weighing100 mg each. Each tablet provides 8 mg of the active agent per unit dosesuitable for oral administration.

Tablet Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof (10 g) is thoroughly blended with cornstarch (50 g),croscarmellose sodium (25 g), lactose (110 mg), and magnesium stearate(5 mg). The mixture is then compressed on a tablet press to form tabletsweighting 200 mg each. Each tablet provides 10 mg of the active agentper unit dose suitable for oral administration.

Capsule Oral Solid Dosage Form

A compound of the invention or a pharmaceutically-acceptable saltthereof is combined with microcrystalline cellulose, polyvinylpyrrolidone, and crosscarmellose sodium in a ratio of 4:5:1:1 by wetgranulation and loaded into gelatin or hydroxypropyl methylcellulosecapsules to provide a unit dosage of, for example, 4 mg, 10 mg or 20 mgactive agent per capsule.

Powder in Capsules

A compound of the invention or a pharmaceutically-acceptable saltthereof (1 to 50 mg) is filled into an empty hydroxypropylmethylcellulose (HPMC) capsule intended for oral administration.

Liquid Formulation

A compound of the invention or a pharmaceutically-acceptable saltthereof (50 mg) is mixed with and fully dissolved in 100 mL low caloriemixed berry sport drink in a capped bottle. Various volumes of thissolution are measured out to provide different dose levels.

Liquid Formulation

A liquid formulation comprising a compound of the invention (0.1%),water (98.9%) and ascorbic acid (1.0%) is formed by adding a compound ofthe invention to a mixture of water and ascorbic acid.

Enteric Coated Oral Dosage Form

A compound of the invention is dissolved in an aqueous solutioncontaining polyvinyl pyrrolidone and spray coated onto microcrystallinecellulose or sugar beads in a ratio of 1:5 w/w active agent:beads andthen an approximately 5% weight gain of an enteric coating comprising anacrylic copolymer, for example a combination of acrylic copolymersavailable under the tradenames Eudragit-L® and Eudragit-S®, orhydroxypropyl methylcellulose acetate succinate is applied. The entericcoated beads are loaded into gelatin or hydroxypropyl methylcellulosecapsules to provide a unit dosage of, for example, 5 mg active agent percapsule.

Enteric Coated Oral Dosage Form

An enteric coating comprising a combination of Eudragit-L® andEudragit-S®, or hydroxypropyl methylcellulose acetate succinate isapplied to a tablet oral dosage form or a capsule oral dosage formdescribed above.

Utility

The present compounds have been designed to deliver a clinicallyefficacious agent directly to the site of action in gastrointestinaltract for the treatment of gastrointestinal inflammatory diseases, inparticular for the treatment of inflammatory bowel diseases such asulcerative colitis and Crohn's disease. The compounds are also expectedto be useful for the treatment of colitis associated with immunecheckpoint inhibitor therapies. In particular, the glucuronide prodrugsof the invention are designed to take advantage of the abundance ofbacterial β-glucuronide enzyme in the gastrointestinal tract, inparticular in the colon, to release the JAK inhibitor tofacitinibpredominantly in the lower gastrointestinal tract. Further, exemplarycompounds of the invention have been shown to be poorly systemicallyabsorbed, thus minimizing the risk of immunosuppression.

The present prodrug compounds are designed to lack biological activity.For example, the compound of formula 1 has no significant affinity for,or potency at, the Janus kinase (JAK) family of enzymes, non-JAKenzymes, or a range of G-protein coupled receptors, ion channels andtransporters which may be expressed in the gastrointestinal (GI) tractor systemically. Biological activity following administration of thepresent compounds is attributable to generated tofacitinib.

As described in the experimental section below, the present compoundshave been extensively profiled in one or more preclinical assays.Compounds have been shown to decompose in the presence of theβ-glucuronidase present in rat colon feces. For example, the metabolicstability of the compound of formula 1 and the formation of tofacitinibwere investigated in homogenate incubations of intestinal lumen contentisolated from the duodenum, ileum, jejunum and colon of rat. Thecompound exhibited a gradient of increasing turnover from stable induodenum and jejunum content (half-life>60 minutes) to modest turnoverin ileum content (half-life 34 minutes) to rapid turnover in the coloncontent (half-life<5 minutes) with evidence of tofacitinib formation.The gradient of increasing compound turnover reflects the increasingpresence of bacteria from upper GI to lower GI.

The release of tofacitinib from the present compounds upon oral dosinghas been studied in mouse, rat, and cynomolgus monkeys. As described inAssays 4, 5, 9, and 10, below, in all species, the prodrugs exhibited asignificantly higher exposure of tofacitinib in the colon than exposurein plasma. In particular, the release of tofacitinib from the compoundof formula 1 in specific segments of the gastrointestinal tract wasstudied in rat and monkey and compared with the concentration obtainedfrom oral dosing of tofacitinib itself at equivalent doses. Not only didcompound 1 exhibit a significantly higher exposure throughout thegastrointestinal tract than exposure in plasma (for example, ratiosgreater than 500 in rat and between about 60 and 150 in monkey in colonsegments) but also showed an increase in the GI tissue concentration andin the GI tissue to plasma concentration ratio relative to that obtainedfrom oral dosing of tofacitinib itself. For example, in monkey, a five-to seven-fold increase in tofacitinib concentration in cecum, proximal,and distal colon tissue was observed from oral administration of theprodrug as compared with that obtained from oral tofacitinib.

Efficacy of certain compounds of the invention was also tested in theoxazolone-induced colitis model in mice. The compounds of formula 1 and4 demonstrated activity in the oxazolone-induced colitis model in miceat lower oral doses than required by direct administration oftofacitinib to achieve an equivalent effect. In addition, theefficacious doses of the compound of formula 1 are associated withreduced systemic exposure of tofacitinib relative to the systemicexposure obtained from dosing tofacitinib itself at its efficaciousdose. In a model of immunosuppression in mice, compound 1 demonstratedminimal effect of immunosuppression at the same dose required todemonstrate comparable efficacy in the oxazolone model (therapeuticindex>3-fold) whereas tofacitinib is immunosuppressive at a dose lowerthan its efficacious dose (therapeutic index≤0.3).

Accordingly, the glucuronide prodrugs of tofacitinib of the inventionare expected to be useful for the treatment of inflammatory boweldisease, in particular ulcerative colitis. The present compounds arealso expected to be useful for the treatment of Crohn's disease and forthe treatment of colitis associated with immune checkpoint inhibitortherapy, a potentially serious consequence of cancer immunotherapies.Immune checkpoint inhibitor therapies include, but are not limited to,cytotoxic T lymphocyte associated antigen 4 (CTLA-4) inhibitors, such asipilimumab (Yervoy®) and tremelimumab; programmed cell death 1 (PD-1)inhibitors, such as pembrolizumab (Keytruda®) and nivolumab (Opdivo®);and programmed death ligand 1 (PD-L1) inhibitors, such as atezolizumab(Tecentriq®), durvalumab, and avelumab. In particular, the compounds areexpected to be useful for the treatment of CTLA-4 inhibitor-inducedcolitis. The compounds may also find utility in the treatment ofadditional conditions such as the gastrointestinal adverse effects ingraft versus host disease, celiac sprue, microscopic colitis, pouchitis,and autoimmune enteropathy.

In one aspect, therefore, the invention provides a method of treating agastrointestinal inflammatory disease in a mammal (e.g., a human), themethod comprising administering to the mammal atherapeutically-effective amount of a compound of the invention or of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and the compound of the invention.

In one embodiment, the gastrointestinal inflammatory disease isulcerative colitis. In another embodiment, the gastrointestinalinflammatory disease is Crohn's disease. And in another embodiment, thegastrointestinal inflammatory disease is colitis associated with immunecheckpoint inhibitor therapy.

The invention further provides a method of treating ulcerative colitisin a mammal, the method comprising administering to the mammal atherapeutically-effective amount of a compound of the invention or of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of the invention.

When used to treat ulcerative colitis, the compound of the inventionwill typically be administered orally in a single daily dose or inmultiple doses per day, although other forms of administration may beused. The amount of active agent administered per dose or the totalamount administered per day will typically be determined by a physician,in the light of the relevant circumstances, including the condition tobe treated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

Suitable doses for treating ulcerative colitis and othergastrointestinal inflammatory disorders are expected to range from about2 to about 60 mg/day of the compound of formula (I), including fromabout 4 to about 50 mg/day and from about 4 to about 40 mg per day foran average 70 kg human.

Combination Therapy

Compounds of the invention may also be used in combination with one ormore agents which act by the same mechanism or by different mechanismsto effect treatment of gastrointestinal inflammatory disorders. Usefulclasses of agents for combination therapy include, but are not limitedto, aminosalicylates, steroids, systemic immunosuppressants, anti-TNFαantibodies, anti-alpha4 (anti-VLA-4) antibodies, anti-integrin α₄β₇antibodies, anti-bacterial agents, and anti-diarrheal medicines.

Aminosalicylates that may be used in combination with the presentcompounds include, but are not limited to, mesalamine, olsalazine andsulfasalazine. Examples of steroids include, but are not limited to,prednisone, prednisolone, hydrocortisone, budesonide, beclomethasone,and fluticasone. Systemic immunosuppressants useful for treatment ofinflammatory disorders include, but are not limited to cyclosporine,azathioprine, methotrexate, 6-mercaptopurine, and tacrolimus. Further,anti-TNFα antibodies, which include, but are not limited to, infliximab,adalimumab, golimumab, and certolizumab, may be used in combinationtherapy. Useful compounds acting by other mechanisms include anti-alpha4antibodies, such as natalizumab, anti-integrin α₄β₇ antibodies, such asvedolizumab, anti-bacterial agents, such as rifaximin, andanti-diarrheal medicines, such as loperamide. (Mozaffari et al. ExpertOpin. Biol. Ther. 2014, 14, 583-600; Danese, Gut, 2012, 61, 918-932; Lamet al., Immunotherapy, 2014, 6, 963-971.)

In another aspect, therefore, the invention provides a therapeuticcombination for use in the treatment of gastrointestinal inflammatorydisorders, the combination comprising a compound of the invention andone or more other therapeutic agents useful for treatinggastrointestinal inflammatory disorders. For example, the inventionprovides a combination comprising a compound of the invention and one ormore agents selected from aminosalicylates, steroids, systemicimmunosuppressants, anti-TNFα antibodies, anti-alpha4 antibodies,anti-integrin α₄β₇ antibodies, anti-bacterial agents, and anti-diarrhealmedicines. Secondary agent(s), when included, are present in atherapeutically effective amount, i.e. in any amount that produces atherapeutically beneficial effect when co-administered with a compoundof the invention.

Further, in a method aspect, the invention provides a method of treatinggastrointestinal inflammatory disorders, the method comprisingadministering to the mammal a compound of the invention and one or moreother therapeutic agents useful for treating gastrointestinalinflammatory disorders.

When used in combination therapy, the agents may be formulated in asingle pharmaceutical composition, as disclosed above, or the agents maybe provided in separate compositions that are administeredsimultaneously or at separate times, by the same or by different routesof administration. When administered separately, the agents areadministered sufficiently close in time so as to provide a desiredtherapeutic effect. Such compositions can be packaged separately or maybe packaged together as a kit. The two or more therapeutic agents in thekit may be administered by the same route of administration or bydifferent routes of administration.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention.

In the examples below, the following abbreviations have the followingmeanings unless otherwise indicated. Abbreviations not defined belowhave their generally accepted meanings.

-   -   Ac=acetyl    -   ACN=acetonitrile    -   alloc=allyloxycarbonyl    -   d=day(s)    -   DCM=dichloromethane    -   DIPEA=N,N-diisopropylethylamine    -   DMAP=4-dimethylaminopyridine    -   Et₃N=triethylamine    -   EtOAc=ethyl acetate    -   EtOH=ethanol    -   h=hour(s)    -   IPA=isopropyl alcohol    -   MeOH=methanol    -   min=minute(s)    -   RT=room temperature    -   tBu=tert-butyl    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran

Reagents and solvents were purchased from commercial suppliers(Sigma-Aldrich, Fluka, etc.), and used without further purification.Progress of reaction mixtures was monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry. Reaction mixtures were worked up as describedspecifically in each reaction; commonly they were purified by extractionand other purification methods such as temperature-, andsolvent-dependent crystallization, and precipitation. In addition,reaction mixtures were routinely purified by column chromatography or bypreparative HPLC, typically using C18 or BDS column packings andconventional eluents. Typical preparative HPLC conditions are describedbelow.

Characterization of reaction products was routinely carried out by massspectrometry and analytical HPLC. Mass spectrometric identification ofcompounds was performed by an electrospray ionization method (ESMS) withan Applied Biosystems (Foster City, Calif.) model API 150 EX instrumentor a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Preparative HPLC Conditions

-   Column: C18, 5 μm. 21.2×150 mm or C18, 5 μm 21×250 or C14, 5 μm    21×150 mm-   Column temperature: Room Temperature-   Flow rate: 20.0 mL/min-   Mobile Phases: A=Water+0.05% TFA    -   B=ACN+0.05% TFA,-   Injection volume: (100-1500 μL)-   Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C18 column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Analytical HPLC Conditions

Method A

Instrument: Agilent 1260 HPLC

Column: LUNA C18 (2), 150×4.60 mm, 3 micron

Column temperature: 35° C.

Flow rate: 1.2 mL/min

Injection volume: 5 μL

Sample preparation: Dissolve in 1:1 ACN:water to ˜0.5 mg/mL solution

Mobile Phases: A=Water:ACN:TFA (98:2:0.05)

-   -   B=Water:ACN:TFA (30:70:0.05)    -   Detector wavelength: 230 nm        Gradient: 28 min total (time (min)/% B): 0/10, 20/100, 22/100,        23/10, 28/10        Method B        Instrument: Agilent 1260 HPLC        Column: Zorbax-Bonus RP C14, 30×2.1 mm, 1.8 micron        Column temperature: 60° C.        Flow rate: 1.2 mL/min        Injection volume: 3 μL        Sample preparation: Dissolve in 1:1 ACN:water to ˜1.0 mg/mL        solution        Mobile Phases: A=Water:TFA (99.9%:0.1%)    -   B=ACN:TFA (99.9%:0.1%)        Detector wavelength: 214 nm        Gradient: 3.0 min total (time (min)/% B): 0/5, 1.5/65, 1.8/95,        2.1/95, 2.5/5, 3.0/5

Preparation 1(2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (10)

(a)(2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (9)

To a 2 L 3-neck flask equipped with a mechanical stirrer was added(2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (8) (51 g, 128.4 mmol), 4-hydroxy-3-nitrobenzaldehyde (20.98g, 125.5 mmol), and silver oxide (37.7 g, 162.7 mmol), followed by ACN(750 mL). The reaction mixture was stirred in the dark for 18 h andfiltered through diatomaceous earth (Celite®). The solid was washed withACN (3×100 mL) and the filtrate was distilled under reduced pressure to100 mL. To the filtrate was added EtOAc (1750 mL) and sat. sodiumbicarbonate (1 L) and the reaction mixture was stirred at RT for 30 min,filtered through Celite and allowed to settle. The organic layer waswashed with sat. sodium bicarbonate (1 L) and brine (1 L), dried oversodium sulfate (100 g) for 2 h, filtered and distilled under reducedpressure to dryness to provide crude Compound 9 as a yellow solid (55 g,90% yield, 97.4% purity) HPLC Retention time 15.61 min.

(b)(2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (10)

To a 1 L 3-neck flask equipped with a mechanical stirrer was addedCompound 9 (32.7 g, 67.6 mmol) followed by DCM (350 mL) and IPA (70 mL).The reaction mixture was stirred to dissolve the solid and then cooledto 0° C. To the solution was added sodium borohydride (1.54 g, 40.6mmol) in three portions, keeping the temperature below 5° C., and thereaction mixture was stirred at 0° C. for 1 h and slowly poured into icewater (400 mL). To the solution was added DCM (350 mL) and the mixturewas stirred for 30 min, allowed to settle for 30 min and the layers wereseparated. The aqueous layer was back extracted with DCM (100 mL). Thecombined organic layers were washed with brine (500 mL). After 30 min,the layers were separated and the brine layer was back extracted withDCM (100 mL). The combined organic layers were dried over sodium sulfate(50 g) for 2 h, filtered through Celite, and distilled under reducedpressure to dryness. The resulting solid was stirred with 95% denaturedEtOH (130 mL) at 50° C. for 30 min and at RT for 12 h to form acrystalline solid which was washed with EtOH (30 mL) and dried undervacuum at RT for 16 h to provide the title compound as a white solid (21g, 66% yield 98% purity) HPLC Method A Retention time 13.18 min.

Preparation 2:(2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (12)

To a 100 mL flask equipped with a magnetic stirrer was added(2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate(10) (4.86 g, 10 mmol) and carbonyldiimidazole (2.11 g, 13 mmol)followed by DCM (50 mL). The reaction mixture was stirred at RT for 3 hto form a solution of(2S,3R,4S,5S,6S)-2-(4-(((1H-imidazole-1-carbonyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate(11).

To a 250 mL 3-neck flask equipped with a magnetic stirrer was addedN¹,N²-dimethylethane-1,2-diamine (3.09 g, 35 mmol, 3.76 mL) followed byDCM (30 mL). The reaction mixture was cooled to 0° C. and acetic acid(2.1 g, 35 mmol, 2 mL) was added slowly at <5° C. to form a suspension.To the suspension was slowly added the solution of intermediate 11; thereaction mixture was stirred at 0-5° C. for 30 min and then at RT for 3h. DCM (30 mL) and water (60 mL) were added and the reaction mixture wasstirred at RT for 10 min. The organic layer was washed with water (2×60mL), dried over sodium sulfate for 2 h and filtered to provide the titlecompound in solution (˜70% yield), which was stored at 0-4° C. and usedwithout purification. HPLC Method A Retention time 11.04 min.

Preparation 3:(2S,3R,4S,5S,6S)-2-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (14)

To a 1 L 3-neck flask equipped with a magnetic stirrer was added3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile(2) (9.65 g, 31 mmol), bis(4-nitrophenyl) carbonate (12.22 g, 40 mmol),and ACN (240 mL) and the reaction mixture was cooled to 0° C. DIPEA(5.59 g, 43 mmol) was added dropwise and the reaction mixture wasstirred at RT for 4 h and cooled to 0° C. To the cooled reaction mixturewas added a solution of(2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-(((methyl(2-(methylamino)ethyl)-carbamoyl)oxy)methyl)-2-nitrophenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate(12) in DCM (350 mL, 30 mmol) at <10° C. and the reaction mixture wasstirred at 15° C. for 1 h and then quenched with acetic acid (2.5 g, 42mmol). DCM (200 mL) and water (300 mL) were added, the layers wereseparated, and the organic layer was washed with 3% sodium carbonate(2×350 mL) and then with 0.5 M HCl (2×200 mL) and with 10% sodiumchloride (200 mL), and dried over sodium sulfate (50 g) for 5 h,filtered, and concentrated to ˜100 mL. The concentrated solution waspurified by silica gel chromatography (600 g silica column, flow rate 60mL/min, gradient: 40% DCM in EtOAc to 100% EtOAc over 5 min, 100% EtOAcfor 60 min, 3% MeOH to 5% MeOH in EtOAc over 30 min, 5% MeOH in EtOAc tocompletion). Pure fractions were combined and distilled to dryness undervacuum to provide the title compound (21.2 g, 97% purity, 73% yield).HPLC Method A Retention time 13.92 min.

Example 1:(2S,3S,4S,5R,6S)-6-(4-((((2-(4-((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (1)

To a 500 mL 3-neck flask equipped with a magnetic stirrer was added(2S,3R,4S,5S,6S)-2-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate(14) (20.9 g, 22 mmol) and THF (210 mL). The reaction mixture was cooledto 0° C. and 1 M LiOH in water (46 mL, 47 mmol) was added over 20 minand the reaction mixture was stirred for 20 min. A second equal portionof LiOH solution was added over 30 min and the reaction mixture wasstirred for 2 h. Acetic acid (5.6 g, 93 mmol) was added and the reactionmixture was transferred to a 2 L round bottom flask. To the flask wasadded ACN (500 mL) and the mixture was distilled under reduced pressureto remove 600 mL of solvent. The process was repeated twice with thethird distillation continued to dryness. The resulting solid wasdissolved in 2% ACN in water (350 mL) and purified in 90 mL batches byreverse phase chromatography (450 g C-18 silica column, flow rate 40mL/min, Solvent A: 0.5% acetic acid in water; Solvent B: ACN, gradient:110 min total (time (min)/% B): 0/2, 10/2, 100/28, 110/28 followed bywash 90% B. Product fractions were combined, the procedure repeatedthree times, and the combined fractions concentrated to about 600 mL andlyophilized to provide the title compound as a solid (11.2 g, 60% yield,99% purity). HPLC Method A Retention time 7.95 min.

Example 2(2S,3S,4S,5R,6S)-6-(2-amino-4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (3)

(a)(2S,3R,4S,5S,6S)-2-(2-amino-4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate(15)

To a solution of(2S,3R,4S,5S,6S)-2-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate(14) (1.55 g, 1.65 mmol) in EtOH (50 mL) was added palladium hydroxideon carbon (11.57 mg, 0.08 mmol). The reaction solution was stirred underhydrogen atmosphere at room temperature for 3 d, filtered through a padof Celite, washed with EtOH, and concentrated in vacuo. The crudematerial was isolated as a brown foam and was used without furtherpurification. (m/z): [M+H]⁺ calcd for C₄₂H₅₃N₉O₁₄ 908.37 found 908.8.

(b)(2S,3S,4S,5R,6S)-6-(2-amino-4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (3)

To a solution of the product of the previous step (0.95 g, 1.05 mmol) ina 1:1:1 mixture of MeOH (3.49 mL), THF (3.49 mL) and water (3.49 mL) wasadded LiOH (63 mg, 2.62 mmol) and the mixture was stirred at roomtemperature for 1 h. The reaction solution was concentrated in vacuo andthe crude material was purified by reverse phase column chromatographyto afford the title compound (96 mg, 12% yield) as a white solid. HPLCMethod B Retention time 0.85 min.

Preparation 4: 4-nitrophenyl4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate(13)

To a solution of3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile(2) (0.75 g, 2.40 mmol) in DCM (12 mL) was added a solution of sodiumhydroxide (0.29 g, 7.20 mmol) in water (4.00 mL) and tetrabutylamoniumbromide (0.08 g, 0.24 mmol). A solution of 4-nitrophenyl chloroformate(0.97 g, 4.80 mmol) in DCM (4 mL) was slowly added. The reaction mixturewas stirred at RT for 1 h, extracted with DCM, and the organic layer waswashed with satd. ammoniun chloride solution and brine, dried oversodium sulfate, filtered and concentrated in vacuo. The crude residuewas purified by column chromatography (0-100% EtOAc in hexanes) toafford title compound (0.94 g, 82%) as a light yellow solid. (m/z):[M+H]⁺ calcd for C₂₃H₂₃N₇O₅ 478.18 found 478.2.

Preparation 5:(2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (18)

(a)(2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (17)

To a solution of(2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate(16) (27.0 g, 61.3 mmol) and Et₃N (25.5 mL, 17.0 mmol) in DCM (250 mL)was slowly added a solution of p-nitrophenyl chloroformate (18.53 g,91.9 mmol) in DCM (100 mL). The solution was stirred at RT for 1 h,diluted with water (100 mL) and extracted with DCM (3×100 mL). Thecombined organic layers were concentrated under reduced pressure and thecrude residue was purified by column chromatography (35% EtOAc inhexanes) to afford the title compound (37.0 g, 56% yield).

(b)(2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (18)

To a solution of(2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate(17) (0.50 g, 0.83 mmol) in DCM (8.26 mL) at RT was addedN′,N²-dimethylethane-1,2-diamine (0.44 mL, 4.13 mmol). After 1.5 h, thereaction solution was filtered and washed with DCM. The filtrate wasconcentrated in vacuo and the residue was purified by columnchromatography (0-10% MeOH in DCM) to afford the title compound (354 mg,77% yield) as a colored solid. (m/z): [M+H]⁺ calcd for C₂₅H₃₄N₂O₁₂555.21 found 555.6.

The following intermediates were prepared by a process analogous toPreparation

Compound R¹ No. methyl 19 (m/z): [M + H]⁺ calcd for C₂₆H₃₆N₂O₁₂ 569.23found 569.7 chloro 20 (m/z): [M + H]⁺ calcd for C₂₅H₃₃ClN₂O₁₂ 591.17found 591.4 methoxy 21 (m/z): [M + H]⁺ calcd for C₂₆H₃₆N₂O₁₃ 585.22found 585.5

Example 3:(2S,3S,4S,5R,6S)-6-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (4)

(a)(2S,3R,4S,5S,6S)-2-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)-(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate(22)

To a solution of(2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (18) (0.35 g, 0.63 mmol) and 4-nitrophenyl4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate(13) (0.30 g, 0.63 mmol) in DMF (5.05 mL) was added Et₃N (0.13 mL, 0.95mmol) and the resulting mixture was stirred at 40° C. After 1.5 h, LC/MSindicated clean product formation. The reaction solution was cooled toroom temperature and concentrated in vacuo. The crude product wasisolated as a yellow residue which was used without furtherpurification. (m/z): [M+H]⁺ calcd for C₄₂H₅₂H₈O₁₄ 893.36 found 893.9.

(b)(2S,3S,4S,5R,6S)-6-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (4)

To a solution of the product of the previous step (0.56 g, 0.63 mmol) ina 1:1:1 mixture of MeOH (2.10 mL), THF (2.10 mL) and water (2.10 mL) wasadded LiOH (40 mg, 1.70 mmol) and the solution was stirred at RT,concentrated in vacuo and the crude residue was purified by reversephase column chromatography to afford the title compound (0.12 g, 27%)as a white solid. (m/z): [M+H]⁺ calcd for C₃₅H₄₄H₈O₁₁ 753.31 found753.8. HPLC Method B Retention time 0.98 min.

Example 4:(2S,3S,4S,5R,6S)-6-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-methylphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (5)

(a)(2S,3R,4S,5S,6S)-2-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)-(methyl)carbamoyl)oxy)methyl)-2-methylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate(23)

To a solution of(2S,3S,4S,5R,6S)-2-(methoxycarbonyl)-6-(2-methyl-4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate(19) (0.46 g, 0.81 mmol) in DCM (8.00 mL) was slowly added a solution of4-nitrophenyl4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate(13) (0.39 g, 0.81 mmol) in DCM (3.00 mL) and the solution was stirredat RT for 1.5 h, concentrated in vacuo and the yellow solid was usedwithout further purification. (m/z): [M+H]⁺ calcd for C₄₃H₅₄H₈O₁₄ 907.38found 907.6.

(b)(2S,3S,4S,5R,6S)-6-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-methylphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (5)

To an ice-cold solution of the product of the previous step (0.73 g,0.81 mmol) in MeOH (29 mL) was slowly added an aqueous solution of LiOH(4.04 mL, 4.04 mmol). After stirring at 0° C. for 1.5 h, the reactionsolution was adjusted to pH 5-6 by the slow addition of 1 N HCl. Thereaction solution was concentrated in vacuo and the crude material waspurified by reverse phase column chromatography to afford the titlecompound (0.47 g, 76% yield) as a white solid. (m/z): [M+H]⁺ calcd forC₃₆H₄₆H₈O₁₁ 767.33 found 767.8. HPLC Method B Retention time 1.02 min.

Example 5:(2S,3S,4S,5R,6S)-6-(2-chloro-4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (6)

(a)(2S,3R,4S,5S,6S)-2-(2-chloro-4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (24)

To a solution of(2S,3R,4S,5S,6S)-2-(2-chloro-4-(((methyl(2-(methylamino)ethyl)-carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (20) (0.33 g, 0.57 mmol) and 4-nitrophenyl4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate(13) (0.27 g, 0.57 mmol) in DMF (4.52 mL) was added Et₃N (0.12 mL, 0.85mmol) and the resulting solution was stirred at 40° C. After 1 h, thesolution was concentrated in vacuo. The product was isolated as a yellowresidue which was used without further purification. (m/z): [M+H]⁺ calcdfor C₄₂H₅₁ClN₈O₁₄ 927.32 found 927.7.

(b)(2S,3S,4S,5R,6S)-6-(2-chloro-4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (6)

To a solution of(2S,3R,4S,5S,6S)-2-(2-chloro-4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (0.52 g, 0.57 mmol) in a 1:1:1 mixture of MeOH (1.88 mL), THF(1.88 mL) and water (1.88 mL) was added LiOH (40 mg, 1.53 mmol) and thesolution was stirred at room temperature for 2 h. The reaction solutionwas concentrated in vacuo and the crude material was purified by reversephase column chromatography to afford the final product (0.22 g, 49%) asa white solid. (m/z): [M+H]⁺ calcd for C₃₅H₄₃ClN₈O₁₁ 787.27 found 787.8.HPLC Method B Retention time 1.04 min.

Example 6:(2S,3S,4S,5R,6S)-6-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (7)

(a)(2S,3R,4S,5S,6S)-2-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-methoxyphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (25)

To a solution of(2S,3R,4S,5S,6S)-2-(2-methoxy-4-(((methyl(2-(methylamino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (21) (0.60 g, 1.02 mmol) in DCM (10.00 mL) was slowly added asolution of 4-nitrophenyl4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate(13) (0.49 g, 1.02 mmol) in DCM (3.00 mL) and the resulting mixture wasstirred at RT for 1.5 h. The reaction solution was concentrated in vacuoand the resulting yellow solid was used without further purification.(m/z): [M+H]⁺ calcd for C₄₃H₅₄H₈O₁₅ 923.37 found 924.1.

(b)(2S,3S,4S,5R,6S)-6-(4-((((2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)(methyl)carbamoyl)oxy)methyl)-2-methoxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylicacid (7)

To an ice-cold solution of the product of the previous step (0.94 g,1.02 mmol) in MeOH (36.80 mL) was slowly added an aqueous solution ofLiOH (5.12 mL, 5.12 mmol). The reaction mixture was stirred at 0° C. for2 h before the pH of the solution was adjusted to 5-6 by slow additionof 1 N HCl. The mixture was concentrated in vacuo and the crude residuewas purified by reverse phase column chromatography to afford the titlecompound (0.46 g, 57% yield) as a white solid. (m/z): [M+H]⁺ calcd forC₃₆H₄₆H₈O₁₂ 783.32 found 783.8. HPLC Method B Retention time 0.99 min.

Example C-1

(a) Compound C-5

To a flask were added the TFA salt of compound C-3 (285 mg, 0.497 mmol)and Compound C-4 (445 mg, 1.289 mmol) followed by DMF (2.485 mL) andtriethylamine (0.416 mL, 2.98 mmol). The next day the reaction mixturewas concentrated by rotary evaporation and purified by normal phasecolumn chromatography (0-15% MeOH in DCM over 15 min, then continuous15% over 5 min) to provide the title compound (90 mg).

(b) Compound C-1

The product of the previous step (45 mg, 0.061 mmol) and potassiumcarbonate (33.8 mg, 0.244 mmol) were dissolved in MeOH (0.8 mL) andwater (0.4 mL) and stirred at RT. After 2 h, MeOH was added to form anazeotrope with water, and DCM (0.4 mL) was added followed by TFA (0.4mL, 5.19 mmol). The reaction mixture was concentrated by rotaryevaporation, dissolved in 1:1 water:ACN, filtered, and purified bypreparative HPLC to provide the title compound (17.7 mg, 97% purity) asan off-white powder. (m/z): [M+H]⁺ calcd for C₂₆H₃₀N₄O₁₅ 639.17 found639.2.

Preparation of Compound C-7

To a solution of compound C-6 (300 mg, 0.374 mmol) dissolved in DCM(3.74 mL) at 0° C. was added N,N-dimethylethylenediamine (159 μl, 1.495mmol) in one portion. The reaction mixture was stirred at 0° C. for 30min, diluted in DCM (15 mL) and washed with water (3×10 mL). The organiclayer was washed with brine, separated, dried over sodium sulfate andfiltered to provide the title intermediate as a yellow oil which wasused without further purification. (m/z): [M+H]⁺ calcd for C₃₃H₄₁H₃O₁₇752.24 found 752.4.

Preparation of Compound C-8

To a solution of sodium hydride (36.4 mg, 0.9105 mmol) dissolved in amixture of THF (3.372 mL) and DMF (1.686 mL) was addedbis(2,4-dinitrophenyl)carbonate (299 mg, 0.759 mmol). The reactionmixture was stirred at 0° C. for 1 h and then a solution of2-(5-fluoro-4-methylpyridin-3-yl)-5-(4-methyl-6-(methylsulfonyl)pyridin-3-yl)-1H-indole(compound C-2M) (200 mg, 0.506 mmol) dissolved in THF (1.2 mL) wasslowly added and the reaction mixture was warmed up to RT. After 9 h,saturated sodium bicarbonate was added and the reaction mixture waswashed with brine. The organic layer was separated, dried over sodiumsulfate, filtered and concentrated. The crude residue was purified bycolumn chromatography (eluted with 0-80% EtOAc in hexanes) to providethe title intermediate (109 mg) as a brown colored oil. (m/z): [M+H]⁺calcd for C₂₈H₂₀FN₅O₈S, 606.10 found 606.3.

Example C-2

(a) Compound C-9

To a solution of compound C-8 (250 mg, 0.413 mmol) in DCM (4.13 mL) wasadded compound C-9 (310 mg, 0.413 mmol), followed by4-dimethylaminopyridine (22 mg, 0.180 mmol). The reaction mixture wasstirred at 40° C. for 1 h, cooled to RT and concentrated to provide thetitle intermediate which was used directly in the next step withoutpurification. (m/z): [M+H]⁺ calcd for C₅₅H₅₇FN₆O₂₀S, 1173.33 found1173.4.

(b) Compound C-2

To a degassed solution of the product of the previous step (485 mg,0.413 mmol) dissolved in THF (4.13 mL) was addedtetrakis(triphenylphosphine)palladium(0) (47.7 mg, 0.041 mmol) andmorpholine (360 μL 4.13 mmol). The reaction mixture was stirred at RTfor 45 min and then water was added and the layers were separated. Theorganic layer was dried over sodium sulfate, filtered and concentratedunder vacuum. The crude residue was purified by preparative HPLC andlyophilized to provide the title compound (136 mg, 99% purity) as awhite powder. (m/z): [M+H]⁺ calcd for C₄₀H₄₁FN₆O₁₄S, 881.24 found 881.2.

Biological Assays

The compounds of the invention have been characterized in one or more ofthe following biological assays. In the assay descriptions, the compoundof formula 1 may alternatively be referenced as compound 1 and similarlyfor the additional compounds of the invention.

Assay 1: Biochemical JAK Kinase Assay

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl₂, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

Serially diluted compounds were pre-incubated with each of the four JAKenzymes and the substrate in white 384-well microplates (Corning) atambient temperature for 1 h. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. The JAK1 kinase activity was also tested at 1 mMATP concentration. Kinase reactions were allowed to proceed for 1 hourat ambient temperature before a 10 μL preparation of EDTA (10 mM finalconcentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies,2 nM final concentration) in TR-FRET dilution buffer (Life Technologies)was added. The plates were allowed to incubate at ambient temperaturefor 1 h before being read on the EnVision reader (Perkin Elmer).Emission ratio signals (520 nm/495 nm) were recorded and utilized tocalculate the percent inhibition values based on DMSO and backgroundcontrols.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC50)and subsequently converted to pK_(i) (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation. Table 1 summarizesresults for compound 1 and tofacitinib (compound 2).

TABLE 1 Enzymatic Potency Property Compound 1 Tofacitinib JAK1 (pK_(i))<6.3 9.1 JAK2 (pK_(i)) <6.2 9.2 JAK3 (pK_(i)) <6.8 9.5 TYK2 (pK_(i)) <67.9 JAK1 (pIC₅₀ at 1 mM ATP) <5 7.7

Assay 2: Metabolic Stability of Compound 1 in β-Glucuronidase from E.Coli

To characterize the intermediate metabolites and final product ofcompound 1 in the presence of β-glucuronidase enzyme, compound 1 (30 μMin DMSO) was incubated at 37° C. in the presence of purifiedβ-glucuronidase from E. coli (100 Units/mL in a 0.1 M potassiumphosphate buffer) over a time course of 0-90 minutes. The incubationswere quenched at timepoints 0, 1, 2, 3, 5, 10, 15, 30, 60, and 90 min byaddition of 100 μL of ACN. Samples were diluted with water+1% formicacid (4×) and analyzed using a Thermo Q-Exactive™ LC-MS system. Compound1 and tofacitinib concentrations were quantified by comparison withstandard curves determined using the same dilutions as the samples.

Rapid disappearance of compound 1 (half-life<5 min) was accompanied withthe rapid and transient formation of the aglycone intermediate (compoundb in Scheme 2). The conversion of the aglycone intermediate to itssubsequent diamine intermediate (compound d) and ultimate activemetabolite (tofacitinib) was observed to be the slower rate-determiningstep. The concentration of tofacitinib was observed to increasegradually over the 90 min time course of the experiment to a finalconcentration of about 20 μM.

To demonstrate the observed conversion of compound 1 to tofacitinib isdue to glucuronidase enzyme interaction, compound 1 (30 μM) wasincubated with β-glucuronidase (45 Units/mL) and the known bacterialβ-glucuronidase inhibitor amoxapine (100 μM). After a 60 min incubation,the final concentration of tofacitinib was 1 μM in the presence of theinhibitor as compared with 7 μM (no inhibitor).

Assay 3: Metabolic Stability in Homogenates Prepared from Rat Upper andLower Intestinal Content

The conversion of compound 1 to tofacitinib was evaluated in theintestinal lumen content prepared from the various GI segments isolatedfrom freshly sacrificed rats. Each segment of content from the duodenum,jejunum, ileum, and colon was diluted 1:10 in Dulbecco's phosphatebuffer saline (DPBS) solution. A 10 mM DMSO stock of compound 1 wasdiluted into DPBS to yield a final substrate concentration of 10 μM. Theincubation was conducted in a water bath at 37° C. and time points weretaken at 0, 5, 10, 20, 40 and 60 min. The total incubation volume was400 μL and 40 μL aliquots were taken at each time point and diluted into160 μL of 97% ACN+3% formic acid+an internal standard. The samples werecentrifuged at 2200 rcf for 10 min and 50 μL of supernatant was dilutedinto 150 μL of water+1% formic acid. The samples were analyzed on an API4000 mass spectrometer for compound 1 and tofacitinib. The half-lifedetermined for the disappearance of compound 1 is summarized below.

TABLE 2 Rat Intestinal Content Compound 1 half-life Duodenum >60 minJejunum >60 min Ileum  34 min Colon  <5 min

Assay 4: Oral Pharmacokinetics of Compound 1 in Rat

The objective of this study was to compare the gastrointestinal mucosaland plasma pharmacokinetics of compound 1 and tofacitinib following asimultaneous oral dose. Male Sprague Dawley rats (n=3/time point) weredosed via oral gavage with 3 mg/kg of compound 1 and a dose normalized1.2 mg of trideuterium labeled tofacitinib (D₃-tofacitinib) formulatedas a solution in 5% DMSO+1% hydroxypropyl methyl cellulos in water. Ateach time point (0.5, 1, 3, 6, 8 and 24 h), plasma samples were taken bycardiac puncture and the following tissues were collected: stomach,upper gastrointestinal tract (sectioned approximately into thirds [U-1,U-2, U-3]). cecum, and lower gastrointestinal tract (sectionedapproximately into halves [L-1, L-2]). Each tissue sample was rinsedwith water, patted dry, transferred to a tared container, weighed,diluted with 3 times the weight of tissue by volume (w/v) with acidifiedwater, homogenized at 6500 RPM (3×45 sec), and frozen. Concentrations oftofacitinib released from compound 1 and of D₃-tofacitinib in eachtissue sample were determined as follows. The tissue samples werevortexed, combined with a 50 μL aliquot of rat plasma, extracted with200 μL of ACN containing an internal standard and quantified against theinternal standard by LC-MS. Concentrations of tofactinib released fromcompound 1 were measureable in plasma between 3 and 8 hours, in thestomach and sections U-1, U-2, and U-3 through 8 hours, and in thececum, and sections L-1, and L-2 between 3 and 24 hours. Concentrationsof D₃-tofacitinib were measureable in plasma between 0.5 and 8 hours, inthe stomach and sections U-1, U-2, and U-3 through 8 hours, and in thececum, and sections L-1, and L-2 between 3 and 24 hours. The resultingstandard pharmacokinetic parameters, C_(max) (maximum concentration) andAUC (0-t) (area under the curve of concentration vs. time, integrated tothe last time point measured) are reported in Table 3.

TABLE 3 Tofacitinib and D₃-Tofacitinib Concentration Rat CompoundAdministered/Analyte Compound 1/TofacitinibD₃-Tofacitinib/D₃-Tofacitinib C_(max) AUC (0-t) Tissue/Plasma C_(max)AUC (0-t) Tissue/Plasma Sample (μg/mL) (μg * hr/mL) Ratio (μg/mL) (μg *hr/mL) Ratio Plasma 0.009 0.044 0.094 0.21 Stomach 2.83 4.68 106 6.959.41 45 U-1 3.51 6.39 145 5.40 7.01 33 U-2 6.56 11.40 259 3.20 7.06 34U-3 10.40 37.90 859 3.39 11.20 53 Cecum 6.39 71.20 1615 0.85 10.10 48L-1 2.28 25.90 587 0.33 3.98 19 L-2 2.41 23.70 537 0.31 3.33 16

Assay 5: Oral Pharmacokinetics of Compound 1 in Cynomolgus Monkey

The objective of this study was to compare the colonic and plasmapharmacokinetics of compound 1 and tofacitinib following a simultaneousoral dose. Male cynomolgus monkeys (n=1/time point) were dosed via oralgavage with 3 mg/kg of compound 1 and 2.1 mg/kg of trideuterium labeledtofacitinib (D₃-tofacitinib) formulated as a solution in 98.5% pH 6citrate buffer+1% hydroxypropyl methylcellulose+0.5% Tween 20. At eachtime point (0.5, 1, 3, 6, 9 and 24 h), plasma samples were taken fromthe femoral vein and the following tissues were collected: stomach,upper gastrointestinal tract (sectioned approximately into thirds [U-1,U-2, U-3]). cecum, proximal colon, distal colon, and rectum. Each tissuesample was rinsed with water, patted dry, transferred to a taredcontainer, weighed, flash frozen, pulverized, and stored at −70° C. Anapproximately 2 g aliquot was diluted 3 times the weight of tissue byvolume (w/v) with control rat plasma in water, homogenzied, and storedat −70° C. Concentrations of tofacitinib released from compound 1 and ofD₃-tofacitinib in each tissue sample were determined as follows. Thesamples were vortexed, and a 50 μL aliquot of plasma or prepared tissuesample was extracted with 200 μL of ACN containing an internal standardand quantified against the internal standard by LC-MS. Concentrations oftofactinib released from compound 1 were measureable in plasma and alltissue samples between 0.5 and 24 hours. Concentrations ofD₃-tofacitinib were measureable in plasma and stomach between 0.5 and 9hours, and in all other tissue sections between 0.5 and 24 hours. Theresulting standard pharmacokinetic parameters, C_(max) (maximumconcentration) and AUC (0-t) (area under the curve of concentration vs.time, integrated to the last time point measured) are reported in Table4.

TABLE 4 Tofacitinib and D₃-Tofacitinib Concentration Monkey CompoundAdministered/Analyte Compound 1/TofacitinibD₃-Tofacitinib/D₃-Tofacitinib C_(max) AUC (0-t) Tissue/Plasma C_(max)AUC (0-t) Tissue/Plasma Sample (μg/mL) (μg * hr/mL) Ratio (μg/mL) (μg *hr/mL) Ratio Plasma 0.14 0.78 0.35 0.83 Stomach 8.01 10.5 13.4 17.9 17.120.7 U-1 19.6 21.5 27.5 18.5 15.7 19.0 U-2 28.8 40.6 51.9 7.96 11.1 13.4U-3 21.7 55.7 71.1 8.96 16.4 19.9 Cecum 31.7 152 194 4.75 22.8 27.6Proximal 19.5 117 149 3.63 15.9 19.2 colon Distal 14.4 46.7 59.6 3.308.86 10.7 colon Rectum 1.99 26.3 33.6 1.00 7.63 9.24

Assay 6: Mouse Model of Oxazolone-Induced Colitis

Oxazolone-induced colitis is an experimental model that has ahistological resemblance to human ulcerative colitis (Heller et al.Immunology, 2002, 17, 629-638). Adult BALB/C mice (25-28 g, 9-12 weeksof age) from BioNeeds (India) were used in the assay. On day 1, animalswere lightly anesthetized with isoflurane and the hairs between theshoulder were carefully removed before oxazolone (6 mg/mouse, 100 μL 4:1acetone: olive oil formulation) or vehicle solution was slowly appliedfor skin sensitization. Six days after skin sensitization, the mice werefasted overnight, anesthetized with ketamine and xylazine administeredIP, and a 1 mL syringe filled with oxazolone solution, was insertedcarefully ˜3.8 cm into the colon of the mouse. Animals were kept in ahead down position and oxazolone (0.5 mg/50 μL/mouse in 50% ethanol) or50% ethanol/saline was rectally instilled very slowly over a minute. Themice were held vertically (head down) for another minute to ensure thatthe entire oxazolone solution remained inside the colon. Drug treatment(PO, BID or TID) or vehicle was initiated the evening prior to theoxazolone intrarectal (IR) challenge. On both first (Day 1) and second(Day 2) days post-oxazolone IR challenge, the Disease Activity Index(DAI) was assessed by treatment-blinded experimenters for each mouse,according to the following subscores: stool consistency (0, normal; 2,loose; 4, diarrhea), gross bleeding and hemoccult test (0, absence; 2,blood tinged; 4, overt blood presence), and weight loss (0, none; 1,1%-5%; 2, 6%-10%; 3, 11%-15%; 4, more than 15%); DAI=average of (stoolconsistency+blood presence+weight loss scores).

An area-under-the-curve (AUC) calculation based on total DAI scores wasperformed to track disease progression during the course of theexperiment. AUC for each experimental group was calculated as: AUC=[(Day1−Day 0)*Average (DAI Score of Day 0 & Day 1)]+[(Day 2−Day 1)*Average(DAI Score of Day 1 & Day 2)]. A Student's t-test compared the DAI AUCscore of the vehicle/vehicle and vehicle/oxazolone groups to evaluatewhether disease was induced following oxazolone treatment. This wasfollowed by a one way ANOVA, with Dunnett's post hoc test, to comparethe DAI AUC score of the vehicle/oxazolone and test compound/oxazolonegroups. Statistical significance was defined by an a level set atp<0.05.

In the oxazolone-induced acute colitis model, the compound of formula 1(3, 10, 30 and 60 mg/kg, PO, BID) produced a significant reversal ofoxazolone-induce colitis, of similar magnitude to that achieved bytofacitinib (30 and 60 mg/kg, PO, BID). In a separate experiment in theoxazolone model, the compound of formula 4 (3, 10, and 30 mg/kg, PO,BID) produced a significant reversal of oxazolone-induce colitis, ofsimilar magnitude to that achieved by tofacitinib (30 mg/kg, PO, BID)

Assay 7: Immunosuppression Effects in Mouse Splenic Natural Killer (NK)Cells

Depletion of mouse splenic cells is an experimental model ofimmunosuppression (Kudlacz et al., Am. J. of Transplantation, 2004, 4,51-57). Compound 1 was assessed in the mouse splenic cell modelfollowing the same treatment paradigm as that used in theoxazolone-induced colitis model (Assay 6).

Adult male Balb/C mice (12-14 weeks of age) from Harlan were used forthe study. Compound 1 (1, 3 and 10 mg/kg, BID) and tofacitinib (10, 30,and 60 mg/kg, BID) were dosed orally for three days to naïve mice.Spleens were harvested 30 min or 3 h post last dose and crushedimmediately for cell subtype staining. Prior to fixation,fluorophore-labelled antibodies for CD19 (FITC; B cells), CD3e (PE; panT cells) and DX5 (APC; NK cells) were incubated with splenocyte samplesfrom each animal to allow for simultaneous, multiple subtype % analysison the flow cytometer. The number of total spleen cells for each animalwas measured by Scepter™ 2.0 Handheld Automated Cell Counter.

The absolute number of lymphocyte subtype population (e.g., splenic B, Tand NK cells) was calculated from the percentage of each subtype timestotal spleen cells for each animal. A one way ANOVA, with Dunnett's posthoc test, was used to compare the splenic lymphocytes number of thevehicle and test compound groups. The α level was set at p<0.05. Datawere presented as the mean±SEM for each group.

Tofacitinib (10, 30 and 60 mg/kg; PO, TID) dose-dependently andsignificantly decreased splenic NK cell counts. In the same study,splenic NK cell counts were unaffected by compound 1 at PO (BID) dosesup to 10 mg/kg (the maximum dose tested). No treatment effect wasobserved for the B and T cell populations with either compound.

This data, in conjunction with the anti-colitic effect of compound 1 inthe mouse model of oxazolone-induced colitis (Assay 6), allow afunctional therapeutic index to be computed as reported below in Table 5

TABLE 5 Functional Therapeutic Index Systemic functional In vivoefficacy activity (splenic NK Functional (oxazolone colitis)* depletion)therapeutic Compound (mg/kg) (mg/kg) index (fold) Compound 1 3 >10 >3Tofacitinib 30 10 0.3 *Effective dose that exhibits a comparablepharmacological effect in the oxazolone model compared to its vehicle

Assay 8: Rat Colon Fecal Homogenate Stability

The objective of this study was to determine the stability of thepresent compounds in rat colon fecal homogenate, i.e. the half-life fordecomposition in the presence of the β-glucuronidase in rat colon feces.

Following sacrifice of a naïve male rat (˜300 g) by cardiac punctureexsanguination, the colon was ligated and removed to an anaerobicchamber (AS-580, Anaerobe Systems). The fecal content was removed withinthe chamber and was diluted 1:10 (1 gram to 9 mL phosphate buffer) andthen homogenized using a handheld Omni Tissue Master. The fecalhomogenate was centrifuged at 2000 g for 10 min to remove bulk and thesupernatant was removed and used for the incubations.

Test articles and a positive control (sulfasalazine) were prepared as 10mM DMSO stocks. The final substrate concentration of each assay was 10μM (30 μM for compound 5). Reactions were started by adding a 5 μLaliquot of diluted test compound stock into 300 μL of rat fecalsupernatant-homogenate. At 0, 15, 30, 60, 90, and 120 min post reactioninitiation, a 50 μL aliquot was removed into 200 μL of acetonitrile with3% formic acid and an internal standard molecule. All samples werecentrifuged at 2000 g for 10 min after which 50 μL of supernatant wasdiluted into 150 μL of water for analysis on an LC-MS system. In vitrohalf-lives for loss of pro-drug were calculated as follows:(T½=0.693/elimination rate constant).

TABLE 6 In vitro colonic stability Compound No. T½ (min) 1 1-15(Multiple values) 4 1-13 (Multiple values) 5 6 6 7 7 3-8  (Multiplevalues)

Assay 9: Oral Pharmacokinetics in Mouse

The object of this study was to assess the plasma and colon conversionof prodrugs of the invention to tofacitinib following oral dosing inmice.

Male Balb/c mice (n=2/timepoint) received a single PO oral gavage dose(5 mg/kg in 1:20 mixture of 5% DMSO and 1% HPMC) of test compounds. At 2hr and 6 hr post dosing mice were sacrificed via cardiac punctureexsanguination, resulting blood samples were placed into Microtainer®tubes containing NaF and then placed on ice. Plasma was obtained bycentrifugation (eppendorf centrifuge, 5804R) for 4 min at approximately12,000 rpm at 4° C.

The colons were removed from exsanguinated mice and the colon fecalcontents gently removed. The colons were flushed with saline and patteddry. They were then homogenized in 3× volume of sterile water using aPrecellys homogenizer at approximately 4° C. All samples were stored at−80° C. for later bioanalysis.

Concentrations of tofacitinib released from prodrug in each tissuesample were determined as follows: The plasma and colon homogenatesamples were vortexed, combined with a 50 μL aliquot of rat plasma,extracted with 200 μL of ACN containing an internal standard andquantified against the internal standard by LC-MS. An area under theconcentration curve (AUC_(0-6 hr)) was calculated for plasma and colontest compound and liberated tofacitinib. The key parameter to assesssuitability was tofacitinib colon/plasma AUC ratio.

TABLE 7 Tofacitinib Concentration Mouse Plasma Compound AUC Colon AUCColon/Plasma No. (μg * hr/mL (μg * hr/g) Ratio 1 0.013 7.6 585 3 0.0019.03 9030

Assay 10: Oral Pharmacokinetics in Rat

The object of this study was to assess the plasma and colon conversionof prodrugs of the invention to tofacinib following oral dosing in rats.

Male Sprague Dawley rats (n=2/timepoint) received a single PO oralgavage dose (5 mg/kg in 1:20 mixture of 5% DMSO and 1% HPMC) of testcompounds. At 0.5, 1, 3, 6 and 24 hr post dosing rats were sacrificedvia cardiac puncture exsanguination, resulting blood samples were placedinto Microtainer® tubes containing NaF and then placed on ice. Plasmawas obtained by centrifugation (Eppendorf centrifuge, 5804R) for 4minutes at approximately 12,000 rpm at 4° C.

The colons were removed from exsanguinated rats and the colon contentsgently removed. The colons were flushed with saline and patted dry. Theywere then homogenized in 3× the weight of sterile water using aPrecellys homogenizer at approximately 4° C. All samples were stored at−80° C. for later bioanalysis.

Concentrations of tofacitinib released from prodrug in each tissuesample were determined as follows: The plasma and colon samples werevortexed, combined with a 50 μL aliquot of rat plasma, extracted with200 μL of ACN containing an internal standard and quantified against theinternal standard by LC-MS. An area under the concentration curve(AUC_(0-6 hr)) was calculated for plasma and colon test compound andliberated tofacitinib. The key parameter to assess suitability wastofacitinib colon/plasma AUC ratio.

TABLE 8 Tofacitinib Concentration Rat Plasma Compound AUC Colon AUCColon/Plasma No. (μg * hr/mL (μg * hr/g) Ratio 1 0.11 14.3 130 3 0.0935.9 399 4 0.03 11.8 393 7 0.06 10.8 171

Assay 11: Metabolic Stability of Comparison Compounds in Rat ColonContent

Colonic fecal content was harvested from a naïve male Sprague Dawley ratand was diluted 1:10 (1 gram to 9 mL phosphate buffer) and thenhomogenized using a handheld Omni Tissue Master. Test compounds,comparison compounds C-1 and C-2 as well as the compound of theinvention, compound 1, were prepared as 10 mM DMSO stock solutions anddiluted into phosphate buffer to a final substrate concentration of 10μM. A 5 μL aliquot of diluted test compound was spiked into 3004 of ratcolonic content homogenate to begin the reaction at 37° C. Aliquots (50μL) were removed from the incubation at the following time points (0,15, 50, 85, and 120 min) and added into 200 μL of ACN with 3% formicacid and an internal standard molecule. Standard curves of eachmetabolite, tofacitinib, 5-ASA (compound C-1M), and the CRAC inhibitor(compound C-2M), were prepared using the same matrix and dilutionprocedure as the samples. All samples were centrifuged at 2000×g for 10min after which 50 μL of supernatant was diluted into 1504 of water andanalyzed using a Thermo Q-Exactive LC-MS system. GraphPad Prism andMicrosoft Excel were used to tabulate and plot the data.

Following incubation at a substrate concentration of 10 μM in ratcolonic content the comparison compounds as well as the compound of theinvention all demonstrated rapid loss of parent molecule in theincubation (half-lives<15 min). However, upon measurement of the activemetabolite of each pro-drug, only compound 1 generated measurable levelsof its active metabolite, tofacitinib. A tofacitinib concentration of8.8 μM was measured after 120 min incubation.

In contrast, compounds C-1 and C-2 failed to generate any measurableamounts of their active metabolites (compound C-1M and C-2M,respectively). The time course of metabolite production over 120 min isillustrated in FIG. 1.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A compound of formula (I):

wherein n is 0, 1 or 2; R¹ is selected from hydrogen, C₁₋₄ alkyl, C₁₋₃alkoxy, amino, nitro, halo, cyano, hydroxy, and trifluromethyl; each R²,when present, is independently selected from C₁₋₄ alkyl, C₁₋₃ alkoxy,amino, nitro, halo, cyano, hydroxyl, and trifluromethyl; R³ is hydrogen,methyl or ethyl; R⁴ is hydrogen, methyl or ethyl; or apharmaceutically-acceptable salt thereof.
 2. The compound of claim 1, ora pharmaceutically-acceptable salt thereof, wherein R¹ is hydrogen. 3.The compound of claim 1, or a pharmaceutically-acceptable salt thereof,wherein R¹ is nitro.
 4. The compound of claim 1, or apharmaceutically-acceptable salt thereof, wherein R¹ is amino.
 5. Thecompound of claim 1, or a pharmaceutically-acceptable salt thereof,wherein n is
 0. 6. The compound of claim 1, or apharmaceutically-acceptable salt thereof, wherein n is
 1. 7. A compoundof formula (II):

wherein R¹ is selected from hydrogen, C₁₋₄ alkyl, C₁₋₃ alkoxy, amino,nitro, halo, cyano, hydroxy, and trifluromethyl; or apharmaceutically-acceptable salt thereof.
 8. The compound of claim 7, ora pharmaceutically-acceptable salt thereof, wherein R¹ is selected fromhydrogen, methyl, methoxy, amino, nitro, and chloro.
 9. The compound ofclaim 7, or a pharmaceutically-acceptable salt thereof, wherein R¹ ishydrogen.
 10. The compound of claim 7, or a pharmaceutically-acceptablesalt thereof, wherein R¹ is amino.
 11. A compound of formula 1:

or a pharmaceutically acceptable salt thereof.
 12. The compound of claim11, wherein the compound is


13. The compound of claim 11, wherein upon contact with aβ-glucuronidase enzyme, a compound of formula 2:

or a salt thereof is produced.
 14. A pharmaceutical compositioncomprising a pharmaceutically acceptable-carrier and a compound of anyone of claims 1, 7, and 11 to 12.